6204 J . Org. Chem., Vol. 64, No. 17, 1999
Kovensky et al.
mmol) in trimethyl phosphite (4 mL) was refluxed for 11 h.
The solvent was removed under reduced pressure, and the
residue was purified by flash chromatography on silica gel (5:4
cyclohexanes-ethyl acetate) affording 5 as a colorless oil (128.0
mg, 76% yield): [R]25D -27 (c 1.0, CHCl3); 1H NMR (200 MHz)
δ 7.36-7.20 (20 H, arom.), 4.76 and 4.63 (two d, 2 H, J ) 11.8
Hz), 4.53-4.20 (m, 7 H), 4.01 (m, 2 H), 3.89 (d, 1 H, J ) 3.7
Hz), 3.85-3.75 (m, 1 H), 3.69 (d, 3 H, J ) 11.9 Hz), 3.68 (d, 3
H, J ) 11.8 Hz), 3.68-3.54 (m, 2 H), 2.27 (ddd, 2 H, J ) 18.2,
6.7, 2.9 Hz); 13C NMR (50 MHz) δ 138.86-127.74, 84.85, 83.39,
83.26, 82.74, 77.71, 75.97, 73.52, 73.28, 71.80, 71.57, 71.18,
24.95 (J C-P ) 141.1 Hz); 31P NMR δ 32.09.
4.85 (ddt, 1 H, J ) 24.8, 6.0 Hz, J 2.6 Hz), 4.56 (dd, 1 H, J )
4.9, 2.6 Hz), 4.45 (dt, 1 H, J ) 4.3, 7.0 Hz), 4.36-4.12 (m, 6
H), 4.00 (dd, 1 H, J ) 8.0, 7.0 Hz), 1.58 (s, 3 H), 1.42 (s, 3 H),
1.24-1.05 (m, 24 H), 0.31, 0.30 and 0.25 (three s, 12 H); 13C
NMR (100 MHz) δ 110.00, 87.56, 81.35, 79.80 (dd, J ) 5.5, 3.2
Hz), 74.96, 65.66, 64.50 (dd, J ) 6.5, 2.5 Hz), 26.41, 25.67,
25.59, 25.58, 16.30, 16.21, -5.43, -5.45, -5.50; 19F NMR δ
-115.2 (ddd, 1 F, J ) 311.4, 101.2, 6.0 Hz), -122.5 (ddd, 1 F,
J ) 311.4, 101.2, 24.8 Hz); 31P NMR δ 6.22 (t, J ) 101.2 Hz).
Anal. Calcd for C26H53F2O8PSi2: C, 50.38; H, 8.62. Found:
C, 50.40; H, 8.67.
C -(1-D e o x y -â-D -g a la c t o fu r a n o s y l)d i flu o r o m e t h -
a n ep h osp h on ic Acid (10). To a solution of 9 (100.0 mg, 0.16
mmol) in dry carbon tetrachloride (2.0 mL) was added trim-
ethylsilyl iodide (342 µL, 2.4 mmol) at 0 °C. After 1 h at room
temperature, the solvent was evaporated, and the residue was
washed with ether. The product was purified by precipitation
(methanol-ethyl acetate) to give 10 (39.3 mg, 81% yield) as a
white foam: [R]25D -20 (c 0.65, H2O); 1H NMR (400 MHz, D2O)
δ 4.45 (t, 1 H, J ) 6.7 Hz), 4.18 (dd, 1 H, J ) 8.2, 6.7 Hz), 4.15
(m, 1 H), 3.85 (dd, 1 H, J )3.4, 8.2 Hz), 3.76 (m, 1 H), 3.66
(dd, 1 H, J ) 11.8, 5.2 Hz), 3.61 (dd, 1 H, J ) 11.8, 7.4 Hz);
13C NMR (100 MHz) δ 81.66, 81.02 (m), 76.08, 75.64, 71.08,
70.37, 62.49; 19F NMR δ -124.2 (dd, 1 F, J ) 87.0, 6.0 Hz),
-124.6 (dd, 1 F, J ) 87.0, 15.0 Hz). FAB+ (matrix triethano-
lamine) 593 [M + (C6H15O3N)2 + 1], 444 (M + C6H15O3N + 1).
Anal. Calcd for C37H43O8P: C, 68.72; H, 6.70. Found: C,
68.22; H, 6.95.
C -(1-D e o x y -r-D -g a la c t o fu r a n o s y l)m e t h a n e p h o s -
p h on ic Acid (6). To a solution of 5 (86.4 mg, 0.13 mmol) in
dry carbon tetrachloride (3.2 mL) was added trimethylsilyl
iodide (0.27 mL, 1.95 mmol) at 0 °C. After 30 min, the solvent
was evaporated, and the residue was washed with ether. The
product was purified by crystallization (ethanol-ethyl acetate)
to give a white solid of mp 121 °C and then dissolved in water
and lyophilized to afford a white hygroscopic solid: [R]25D -29
1
(c 0.42, H2O); H NMR (400 MHz, D2O, as the triethylammo-
nium salt) δ 4.21 (m, 1 H), 4.11 (broad s, 1 H), 4.05 (broad d,
1 H), 3.76 (m, 1 H), 3.69 (dd, 1 H, J ) 4.0 Hz, J ) 11.8 Hz),
3.64 (t, 1 H, J ) 4.9 Hz), 3.59 (dd, 1 H, J ) 7.0, 11.8 Hz), 1.95
(dd, 2 H, J ) 6.7, 18.0 Hz); 13C NMR (50 MHz) δ 83.89, 78.51,
77.96, 77.71, 71.47, 62.58, 27.92 (J C-P ) 131.2 Hz); 31P NMR
δ 25.14. FAB- (matrix glycerol) 256 (M - 1); FAB+ (matrix
triethanolamine) 557 [M + (C6H15O3N)2 + 1], 408 (M +
C6H15O3N + 1).
Ur id in e Dip h osp h a te C-r-D-Ga la ctofu r a n ose (11). The
triethylammonium salt of C-(1-deoxy-R-D-galactofuranosyl)-
methanephosphonic acid (50.0 mg, 0.108 mmol, easily obtained
from 6) was dissolved in anhydrous pyridine (0.5 mL) and
evaporated at reduced pressure. The flask was kept moisture-
free by using nitrogen to bring the pressure back to normal.
This procedure was repeated three times, and the residue was
finally dissolved in pyridine. The solution was added to a
suspension of uridine 5′-monophosphate morpholidate and
4-morpholine-N,N′-dicyclohexylcarboxamidine (100.0 mg, 0.145
mmol, Sigma) in pyridine (0.5 mL). The activated nucleoside
monophosphate was previously dried by coevaporating dry
pyridine three times. After coevaporation with pyridine two
more times, 1.5 mL of pyridine was added, and the mixture
was stirred under nitrogen for 5 days. The product was
concentrated, dissolved in a minimum amount of water, and
applied to a Dowex 1 column (HCO3-; 15 × 1 cm, 200-400
mesh). The column was eluted with a NH4HCO3 gradient (0.0-
0.5 M). The appropriate fractions were pooled and lyophilized.
Water was added to the residue, and the solution was
lyophilized again. This procedure was repeated three times.
The ammonium salt of UDP-C-Galf was obtained as a white
powder (52.0 mg, 80% yield): 1H NMR (400 MHz, D2O) δ 7.87
(d, 1 H, J ) 8.1 Hz), 5.91 (d, 1 H, J ) 4.6 Hz), 5.87 (d, 1 H, J
) 8.1 Hz), 4.29 (t, 1 H, J ) 4.9 Hz), 4.25 (t, 1 H, J ) 4.9 Hz),
4.19 (m, 1 H), 4.17-4.10 (m, 2 H), 4.05-3.92 (m, 1 H), 3.72
(m, 1 H), 3.65 (dd, 1 H, J ) 4.0, 11.8 Hz), 3.62 (m, 2 H), 3.58-
3.52 (m, 2 H), 1.81 (m, 2 H); 13C NMR (100 MHz) δ 160.69,
160.36, 141.87, 103.97, 89.11, 83.90, 83.52 (d, J ) 7.9 Hz),
79.38, 79.03, 78.88, 74.12, 71.86, 69.94, 67.29 (d, J ) 7.3 Hz),
63.12, 29.57 (d, J ) 123.4 Hz); 31P NMR δ 5.16, 14.12;
2,5-An h yd r o-1-d eoxy-1,1-d iflu or o-3,4-(bis-O-ter t-bu tyl-
d im eth ylsilyl)-6,7-O-isop r op ylid en e-D-ga la cto-h ep t-1-en -
itol (8). To a solution of 2,3-(bis-O-tert-butyldimethylsilyl)-5,6-
O-isopropylidene-D-galactono-1,4-lactone 710 (2.0 g, 4.48 mmol)
and dibromodifluoromethane (2.0 mL, 22.8 mmol) in anhy-
drous THF (35 mL) was added tris(dimethylamino)phosphine
(8.3 mL, 45.7 mmol) dissolved in THF (20 mL) at -20 °C, and
the mixture was stirred at room temperature for 30 min. Zinc
powder (1.5 g, 22.8 mmol) and tris(dimethylamino)phosphine
(0.8 mL) were added, and the mixture was heated to reflux
for 15 h. The mixture was cooled to room temperature, and
ether (50 mL) was added. The ether layer was decanted and
the residue washed with ether (50 mL). The combined ether
extracts were washed with saturated copper sulfate solution
until the solution remained blue and then with water and brine
and dried over MgSO4. The solvent was removed under
reduced pressure to give a yellow oil. Flash chromatography
on silica gel (99:1 then 95:5 cyclohexane-ethyl acetate) af-
forded 8 (1.34 g, 62% yield) as a pale yellow oil: [R]25 -26 (c
D
1.0, CHCl3); CI-MS: 498 (M + NH4+), 481 (M + 1); H NMR
1
(250 MHz) δ 4.50-4.34 (m, 2 H), 4.15 (d, 1 H, J ) 8.5 Hz),
4.01 (dd, 1 H, J ) 6.7, 8.0 Hz), 3.80 (broad s, 1 H), 3.75 (t, 1
H, J ) 7.6 Hz), 1.45 and 1.37 (two s, 6 H), 0.90 (s, 18 H), 0.10
and 0.09 (two s, 12 H); 13C NMR (62.5 MHz) δ 150.11 (dd, J )
267, 289 Hz), 121.02 (dd, J ) 13, 50 Hz), 110.12, 91.83, 79.41
(d, J ) 2 Hz), 75.59 (d, J ) 4 Hz), 75.44 (d, J ) 7 Hz), 65.62,
26.94, 26.66, 25.61, 25.56, 25.19, 17.69, 17.64, -459, -4.69,
-5.01, -5.04, -5.28, -5.29; 19F NMR δ -104.4 (d, 1 F, J ) 91
Hz), -119.3 (d, 1 F, J ) 91 Hz).
electrospray+ 583 (M + NH4 + 1).
+
Anal. Calcd for C22H42F2O5Si2: C, 54.96; H, 8.81. Found: C,
54.89; H, 8.77.
In h ibition Stu d ies. The assay mixture (total volume 100
µL) contained 100 µM UDP-Galp, 49 µg of protein (crude E.
coli extract in which M. tuberculosis UDP-galactopyranose
mutase was expressed as described previously13), 2 mM
NADH, 1 mM MgCl2, 50 mM HEPES buffer at pH 7.6, and
with and without UDP-C-Galf to be tested for inhibition
activity. The reactions were incubated for 30 min at 37 °C and
then stopped by the addition of ethanol. The formation of UDP-
Galf was then monitored by HPLC. Under these conditions,
the formation of UDP-Galf was in the linear range.
Diet h yl C-[2,3-(Bis-O-ter t-b u t yld im et h ylsilyl)-5,6-O-
is o p r o p y lid e n e -â-D -g a la c t o fu r a n o s y l]d iflu o r o m e t h -
a n ep h osp h on a te (9). A solution of 8 (500 mg, 1.0 mmol),
freshly distilled diethyl phosphite (536 µL, 4.0 mmol), and tert-
butylperoxypivalate (60 µL, freshly prepared from tert-butyl-
hydroperoxide and pivaloyl chloride) in anhydrous benzene (0.5
mL) was degassed and stirred at 60 °C. After 24 h, the mixture
was cooled to room temperature, and more tert-butylperox-
ypivalate (80 µL) was added. After 24 h at 60 °C, the mixture
was chromatographed on silica gel (5:1 cyclohexane-ethyl
acetate) to give 9 (361.7 mg, 56% yield) as a colorless oil: [R]25
D
(13) Weston, A.; Stern, R. J .; Lee, R. E.; Nassau, P. M.; Monsey, D.;
Martin, S. L.; Scherman, M. S.; Besra, G. S.; Duncan, K.; McNeil, M.
R. Tuberc. Lung Dis. 1998, 78, 123.
-7 (c 1.1, CHCl3); CI-MS: 636 (M + NH4+), 619 (M + 1), 561
1
(M - t-Bu‚); H NMR (400 MHz) δ 5.01 (t, 1 H, J ) 2.6 Hz),